10. Papillomavirus 2 Flashcards
why does the virus prevent the keratinocytes from losing its nuclei?
normally, as keratinocytes differentiate they lose their nuclei
but the virus wants the cell to keep its nuclei so it stays in a pseudo S-phase with DNA replication machinery so the virus can replicate
describe the size and shape of the papillomavirus genome
8 kbp circular dsDNA
how many proteins does the genome encode for?
<10
what are the 3 functional regions of the genome?
- early genes
- late genes
- regulatory region (LCR or URR)
what do LCR and URR stand for?
LCR = long control region
URR = upstream regulatory region
what is the function of regulatory region (LCR and URR)? (3)
- transcription
- DNA replication (for E1 and E2)
- segregation of genome
are the 8 major proteins?
- E1
- E2
- E4
- E5
- E6
- E7
- L1
- L2
what is the only HPV enzyme and its function?
E1 –> DNA helicase
what are the 3 functions of E2?
- helicase loader
- transcriptional repressor
- segregation factor
what are the 3 overall roles of E1 and E2 in the replication cycle?
- viral DNA replication
- gene expression
- segregation
describe E2 acting as a transcriptional repressor
binds LCR and represses transcription of E6 and E7
required because if too much E6 and E7, things go wrong so E2 is a regulator, but then later in cancer cells E2 is not made so oncogenes can be made
what are the functions of E4 and E5 and their role in the replication cycle?
E4: disrupts cytokeratin network
E5: recycles growth factor receptors
have a role in genome amplification
what are the molecular functions of E6 and E7 and their role in the replication cycle?
E6: binds p53
E7: binds pRb
involved in viral oncogenes
what are the roles of L1 and L2?
L1 = major capsid protein
L2 = minor capsid protein
what causes senescence?
once a primary cell divides multiple times, its telomeres shorten at every division until the cell eventually dies
how does HPV prevent senescence?
- E6 activated telomerase to prevent telomere shortening
- E6/E7 inhibit p53/pRb
- cells become IMMORTALIZED
- Ras V12 oncogene activated
- cells become TRANSFORMED
what does it mean for cells to immortalized?
when cells express E6 and E7, they are not tumorigenic but can grow indefinitely
when do cells become tumorigenic? what does it mean for a cell to be tumorigenic?
when immortalized cells are transformed by adding mutations that activate Ras oncogene –> cells have additional growth properties and are tumorigenic
what 2 proteins maintain the viral genome as an episome?
E1 and E2
what assay can be used to examine viral DNA? how is this helpful?
southern blot –> can visualize the presence of an episome, therefore the viral genome and functional and actively replicating
what are organotypic raft cultures?
keratinocytes put at the interface btwn cell culture and air
describe normal keratinocytes in raft cultures
epithelium is healthy, only basal layer is actively proliferating and synthesizing DNA
describe HPV-immortalized keratinocytes in raft cultures
thicker epithelium, some differentiation
all cells maintain nuclei therefore all cells proliferating and making DNA replication machinery due to E6
is cervical cancer rare or common?
rare relative to the high number of high-risk HPV infections
do all high-risk HPV infections lead to cervical cancer?
no, but all cervical cancer is caused by high-risk HPV infection
what is the difference between the viral genome during the normal viral life cycle vs cancers?
during the normal viral life cycle: the HPV genome is maintained as an episome
in cancers: the HPV genome is randomly integrated into the host chromosomes
integration is a biomarker of:
integration is a biomarker of cancer progression
how does viral genome integration allow for cancer progression?
E2 is disrupted –> E2 normally represses E6 and E7 transcription but now there can be higher expression of E6 and E7
what happens when E2 is re-expressed in cervical carcinoma cells?
E6 and E7 are repressed so p53 and pRb are reactivated –> cells cannot proliferate and die by senescence/apoptosis
why are cervical carcinoma cells “addicted” to oncogenes? what does this mean for cancer therapy?
any repression of E6 and E7 = death, so the cells require continuous expression of E6 and E7 for growth
this means E6 and E7 could be targets for cancer therapy
what happens when E7 acts on pRB?
normally, pRB is in complex with E2F but E7 disrupts this complex –> allows E2F to stimulate proliferation (S phase)
what does E2F do?
E2F is a family of transcription factors that regulate DNA replication machinery
what is the result of E7-induced proliferation and how does E6 affect it?
E7-induced proliferation results in apoptosis
how do cells normally progress into S phase?
normally, cyclin-Cdk2 phosphorylates pRb to release it from E2F so cell can undergo proliferation/S phase
what are the 3 mechanisms by which E7 activates E2F?
- E7 binds pRb to induce its degradation by proteasome
- E7 binds and activates Cdk2
- E7 binds and inhibits p21 and p27 (natural inhibitors off cyclin/cdk2
describe the protein structure of E7
small zinc-finger protein with LxCxE motif which binds Rb
how does E6 inhibit p53? and 3 steps
E6 induces the degradation of p53 by the proteasome
- E6 forms complex with E6AP
- complex binds p53 and promotes its poly-ubiquitination
- poly-ubiquitinated p53 is targeted and degraded by proteasome
what type of protein is E6AP?
cellular E3-ubiquitin ligase
is E6AP normally involved in p53 degradation?
no!! the virus repurposed E6AP
what happens if you express E6 in cells?
p53 levels immediately decrease
describe the protein structure of E6
small protein with 2 zinc fingers
describe the binding of E6 to E6AP
E6 binds to alpha helical LxxLL motif (hydrophobic amino acids) in E6AP
telomerase is commonly expressed in what type of cells?
cancer cells
describe the structure and components of telomerase
ribonucleoprotein complex
TER RNA and TERT protein
what is the activity of TERT protein?
reverse transcriptase activity
why does E6 activate telomerase? how?
to sustain cellular proliferation
E6 increases TERT transcription
